Performance of a multigene genomic classifier and clinical parameters in predicting malignancy in a Southeast Asian cohort of patients with cytologically indeterminate thyroid nodules.

BACKGROUND: Most thyroid nodules are benign. It is important to determine the likelihood of malignancy in such nodules to avoid unnecessary surgery. The primary objective of this study was to characterize the genetic landscape and the performance of a multigene genomic classifier in fine-needle aspiration (FNA) biopsies of cytologically indeterminate thyroid nodules in a Southeast Asian cohort. The secondary objective was to assess the predictive contribution of clinical characteristics to thyroid malignancy. METHODS: This prospective, multicenter, blinded study included 132 patients with 134 nodules. Molecular testing (MT) with ThyroSeq v3 was performed on clinical or ex-vivo FNA samples. Centralized pathology review also was performed. RESULTS: Of 134 nodules, consisting of 61% Bethesda category III, 20% category IV, and 19% category V cytology, and 56% were histologically malignant. ThyroSeq yielded negative results in 37.3% of all FNA samples and in 42% of Bethesda category III-IV cytology nodules. Most positive samples had RAS-like (41.7%), followed by BRAF-like (22.6%), and high-risk (17.9%) alterations. Compared with North American patients, the authors observed a higher proportion of RAS-like mutations, specifically NRAS, in Bethesda categories III and IV and more BRAF-like mutations in Bethesda category III. The test had sensitivity, specificity, negative predictive value, and positive predictive value of 89.6%, 73.7%, 84.0%, and 82.1%, respectively. The risk of malignancy was predicted by positive MT and high-suspicion ultrasound characteristics according to American Thyroid Association criteria. CONCLUSIONS: Even in the current Southeast Asian cohort with nodules that had a high pretest cancer probability, MT could lead to potential avoidance of diagnostic surgery in 42% of patients with Bethesda category III-IV nodules. MT positivity was a stronger predictor of malignancy than clinical parameters.

Dynamic altruistic cooperation within breast tumors.

BACKGROUND: Social behaviors such as altruism, where one self-sacrifices for collective benefits, critically influence an organism's survival and responses to the environment. Such behaviors are widely exemplified in nature but have been underexplored in cancer cells which are conventionally seen as selfish competitive players. This multidisciplinary study explores altruism and its mechanism in breast cancer cells and its contribution to chemoresistance. METHODS: MicroRNA profiling was performed on circulating tumor cells collected from the blood of treated breast cancer patients. Cancer cell lines ectopically expressing candidate miRNA were used in co-culture experiments and treated with docetaxel. Ecological parameters like relative survival and relative fitness were assessed using flow cytometry. Functional studies and characterization performed in vitro and in vivo include proliferation, iTRAQ-mass spectrometry, RNA sequencing, inhibition by small molecules and antibodies, siRNA knockdown, CRISPR/dCas9 inhibition and fluorescence imaging of promoter reporter-expressing cells. Mathematical modeling based on evolutionary game theory was performed to simulate spatial organization of cancer cells. RESULTS: Opposing cancer processes underlie altruism: an oncogenic process involving secretion of IGFBP2 and CCL28 by the altruists to induce survival benefits in neighboring cells under taxane exposure, and a self-sacrificial tumor suppressive process impeding proliferation of altruists via cell cycle arrest. Both processes are regulated concurrently in the altruists by miR-125b, via differential NF-κB signaling specifically through IKKß. Altruistic cells persist in the tumor despite their self-sacrifice, as they can regenerate epigenetically from non-altruists via a KLF2/PCAF-mediated mechanism. The altruists maintain a sparse spatial organization by inhibiting surrounding cells from adopting the altruistic fate via a lateral inhibition mechanism involving a GAB1-PI3K-AKT-miR-125b signaling circuit. CONCLUSIONS: Our data reveal molecular mechanisms underlying manifestation, persistence and spatial spread of cancer cell altruism. A minor population behave altruistically at a cost to itself producing a collective benefit for the tumor, suggesting tumors to be dynamic social systems governed by the same rules of cooperation in social organisms. Understanding cancer cell altruism may lead to more holistic models of tumor evolution and drug response, as well as therapeutic paradigms that account for social interactions. Cancer cells constitute tractable experimental models for fields beyond oncology, like evolutionary ecology and game theory.

Pitfalls in Lymph Node Fine Needle Aspiration Cytology.

Background Fine needle aspiration cytology (FNAC) is an accurate, minimally invasive and cost-effective biopsy method for enlarged lymph nodes. While the role of lymph node FNAC in the diagnosis of infectious or reactive conditions and metastatic malignancy is unquestioned, differing views still exist on its role in the diagnosis of lymphoma. Nevertheless, regardless of the practice setting, pitfalls and potential for error exist, and it is incumbent upon the pathologist to be aware of these pitfalls; as this is the first line of defence against errors. Summary This discussion will focus on potential interpretational errors, specifically highlighting scenarios leading to false negative and false positive diagnosis and errors in tumour classification, with an emphasis on cytomorphology. Potential entities that may fly below the radar of the pathologist - so-called "off radar entities" are also discussed, as a reminder to consider broad differentials in cases with unusual morphologic features. Some reasons for false negative diagnoses include low grade lymphomas that mimic a mixed, polymorphous reactive lymphoid population; or aspirates with a paucity of lesional cells, either through sampling error or the intrinsic nature of the entity e.g. nodular lymphocyte predominant Hodgkin lymphoma. Some of the potential causes of false positive diagnoses that are discussed include viral-associated lymphadenopathy, Kikuchi-Fujimoto lymphadenitis or benign adnexal lesions mimicking metastatic malignancy. Errors in tumour classification covered include metastatic carcinoma, sarcoma, melanoma and lymphoma mimicking each other, and Hodgkin lymphoma and its mimics. Finally, less common entities such as follicular dendritic cell sarcoma and others are briefly mentioned, to remind us of conditions that may slip under our diagnostic radar. Key Messages A systematic review of diagnostic pitfalls and traps are elucidated here, with some tips to avoid these traps. The triple approach to the diagnostic workup is emphasised; which includes rigorous clinicopathologic correlation, attention to cytomorphology and judicious application of ancillary tests.

Outcomes of a Phase II Study of Intraperitoneal Paclitaxel plus Systemic Capecitabine and Oxaliplatin (XELOX) for Gastric Cancer with Peritoneal Metastases.

BACKGROUND: Adding intraperitoneal paclitaxel (IP-PTX) to paclitaxel/5-fluoropyrimidine has shown promising results in patients with gastric cancer peritoneal metastases (GCPM) but has not been studied with standard-of-care platinum/fluoropyrimidine combinations. Our goal to was evaluate IP-PTX with capecitabine/oxaliplatin (XELOX) in GCPM. METHODS: Forty-four patients with GCPM received IP PTX (40 mg/m2, Days 1, 8), oral capecitabine (1000 mg/m2 twice daily, Days 1-14) and intravenous oxaliplatin (100 mg/m2, Day 1) in 21-day cycles. Patients with synchronous GCPM underwent conversion surgery if they had good response after chemotherapy, conversion to negative cytology, no extraperitoneal metastasis, and no peritoneal disease during surgery. The primary endpoint was overall survival and secondary endpoints were progression-free survival and safety. Outcomes from the trial were compared against a matched cohort of 39 GCPM patients who received systemic chemotherapy (SC) comprising platinum/fluoropyrimidine. RESULTS: The median OS for the IP and SC groups was 14.6 and 10.6 months (hazard ratio [HR] 0.44; 95% confidence interval [CI], 0.26-0.74; p = 0.002). The median PFS for the IP and SC group was 9.5 and 4.4 months respectively (HR 0.39; 95% CI 0.25-0.66; p < 0.001). Patients in the SC group were younger (IP vs. SC, 61 vs. 56 years, p = 0.021) and had better performance status (ECOG 0, IP vs. SC, 47.7% vs. 76.9%, p = 0.007) compared with the IP cohort. In IP group, conversion surgery was performed in 36.1% (13/36) of patients, with a median OS of 24.2 (95% CI 13.1-35.3) months and 1-year OS of 84.6%. CONCLUSIONS: IP PTX with XELOX is a promising treatment option for GCPM patients. In patients with good response, conversion surgery was feasible with favourable outcomes.

Multi-institutional validation of a modified scheme for subcategorizing salivary gland neoplasm of uncertain malignant potential (SUMP).

BACKGROUND: The salivary gland neoplasm of uncertain malignant potential (SUMP) category in the Milan System is diagnostically challenging. This study aims to validate a modified scheme for subcategorizing SUMP in a large multi-institutional cohort. METHODS: Retrospective review of salivary gland fine-needle aspirations (FNAs) from 10 institutions were classified based on the Milan System. Cases diagnosed as SUMP with available cytology slides and surgical follow-up were retrieved for review and subcategorized based on a modified scheme as follows: basaloid SUMP (B1: absent/scant nonfibrillary matrix; B2: presence of nonfibrillary/mixed-type matrix), oncocytic/oncocytoid SUMP (O1: with mucinous background; O2: without mucinous background), and SUMP not otherwise specified (NOS). RESULTS: A total of 742 (7.5%) cases from 9938 consecutive salivary gland FNAs were classified as SUMP. Among them, 525 (70.8%) had surgical follow-up and 329 (62.7%) were available for review. The overall risk of malignancy (ROM) of SUMP was 40.4%. There were 156 cases (47.4%) subcategorized as basaloid SUMP with a ROM of 36.5%, 101 (30.7%) as oncocytic/oncocytoid SUMP with a ROM of 52.5%, and 72 (21.9%) as SUMP NOS with a ROM of 31.9%. The ROM of oncocytic/oncocytoid SUMP was significantly higher than basaloid SUMP (P = .0142) and SUMP NOS (P = .0084). No significant differences in ROM were noted between B1 and B2 (36.7% vs 36.4%, P = 1.0000) and O1 and O2 (65.2% vs 48.7%, P = .2349). CONCLUSIONS: The ROM of oncocytic/oncocytoid SUMP was 52.5% and significantly higher than that of basaloid SUMP (36.5%, P = .0142) and SUMP NOS (31.9%, P = .0084), whereas no significant differences in ROM were noted for cases with different types of extracellular matrix or background material.

In vivo demonstration of a novel non-invasive model for inducing localized hypothermia to ameliorate hepatotoxicity.

Moderate hypothermia (32 °C) has been previously shown to ameliorate drug-induced liver injuries in vitro. However, there are concerns regarding its clinical relevance as it remains a challenge to perform selective liver cooling in a non-invasive manner. To reconcile this dilemma, we propose the use of pulsed cooling for regional hypothermic conditioning in liver. This involves intermittent cooling applied in pulses of 15 min each, with a one-hour recovery interval between pulses. Cooling is achieved by applying ice packs to the cutaneous region overlying the liver. Through an in vivo C57BL/6NTac mouse study, we demonstrated the feasibility of attaining localized hypothermia close to the liver while maintaining core body temperature. This has successfully ameliorated acetaminophen-induced liver injury based on the liver function tests, liver histology and total weight change. Collectively, we provide a proof of concept for pulsed external localized cooling as being clinically actionable to perform induced selective hypothermia.

Constitutive Cytomorphologic Features of Medullary Thyroid Carcinoma Using Different Staining Methods.

(1) Background: Accurate preoperative identification of medullary thyroid carcinoma (MTC) is challenging due to a spectrum of cytomorphologic features. However, there is a scarcity of studies describing the cytomorphologic features as seen on fine-needle aspiration (FNA) smears prepared using different staining methods. (2) Methods: We performed a retrospective study on MTC cases with available FNA slides from 13 hospitals distributed across 8 Asia-Pacific countries. The differences in the constitutive cytomorphologic features of MTC with each cytopreparatory method were recorded. A comparative analysis of cytologic characteristics was carried out with appropriate statistical tests. (3) Results: Of a total of 167 MTC samples retrospectively recruited, 148 (88.6%) were interpreted as MTC/suspicious for MTC (S-MTC). The staining methods used were Papanicolaou, hematoxylin-eosin, and Romanowsky stains. Seven out of the eleven cytologic criteria can be readily recognized by all three cytopreparatory methods: high cellularity, cellular pleomorphism, plasmacytoid cells, round cells, dyshesive cells, salt-and-pepper chromatin, and binucleation or multinucleation. An accurate diagnosis was achieved in 125 (84.5%) of the 148 samples whose FNAs exhibited five or more atypical features. Conclusions: The present work is the first study on MTC to compare the morphological differences among the cytologic staining techniques. We investigated the constitutive features and the reliability of diagnostic parameters. A feasible scoring system based upon cytomorphologic data alone is proposed to achieve a high degree of diagnostic accuracy.

COVID-19 post-vaccination lymphadenopathy: Report of cytological findings from fine needle aspiration biopsy.

The coronavirus COVID-19 pandemic has spurred the rapid development of vaccines, with vaccination programmes already underway in many countries. Regional lymphadenopathy is one of the documented side effects of vaccination. We document the fine needle aspiration cytological findings of an enlarged supraclavicular lymph node in a 34-year-old Asian female following the first dose of the Pfizer-BioNTech COVID-19 mRNA vaccine, which appears to be the first such report in a premorbidly well patient with no known history of malignancy. The cytological findings featured a reactive pattern in keeping with follicular hyperplasia, with prominent germinal centre elements including lymphohistiocytic aggregates and tingible-body macrophages. Despite an increased proportion of larger lymphocytes, the overall pattern was in keeping with a reactive pattern, bearing in mind the temporal and geographic relation to the vaccination injection. In instances of localised lymphadenopathy, particularly in supraclavicular or axillary locations, pathologists should be cognizant of the possibility of post-vaccination reactive lymphadenopathy, and seek clinical and radiological hints favouring a benign process, whilst recognising potential morphological overlaps with lymphoproliferative disorders. Awareness of this diagnostic pitfall is especially important as COVID-19 vaccination coverage is ramped up worldwide, leading to an expected increase in incidence of post-vaccination reactive lymphadenopathy.

Cytologic diagnosis of medullary thyroid carcinoma in the Asia-Pacific region.

BACKGROUND: The accurate preoperative identification of medullary thyroid carcinoma (MTC) is challenging due to the rarity of tumor and variable cytologic appearance. The Asian experience with diagnosing MTC by fine-needle aspiration (FNA) was scarcely reported. METHODS: Cases of MTC with available FNA slides were enrolled from 13 hospitals representing 8 Asia-Pacific countries. Clinicopathological information, including sample preparation technique, staining method, original cytologic diagnosis and review diagnosis were collected. RESULTS: Of a total of 145 MTC cases retrospectively recruited, 99 (68.3%) were initially interpreted as MTC/suspicious for MTC (S-MTC). The distribution of original FNA diagnostic categories was not associated with the staining method or sample preparation technique. The staining methods used were Papanicolaou, hematoxylin-eosin and Romanowsky stains. Liquid-based cytology (LBC) was used only in three countries. After reviewing all cases, the diagnostic rate of MTC/S-MTC increased to 91.7% (133/145). Cases with initially unrecognized MTC had either marked pleomorphism or cytology mimicking papillary carcinoma or follicular neoplasm. Although LBC provided certain benefits, there was no significant difference in diagnostic accuracy between conventional smear and LBC. Immunocytochemistry was available in 38 cases (26.2%), all of which were correctly recognized as MTC. CONCLUSION: Our report summarizes how MTC is handled in contemporary Asian thyroid FNA practice. Although the detection rate of MTC by cytology alone is less satisfactory, integration with ancillary tests could achieve an excellent performance. The recognition of constitutive cytomorphologic features is needed for each cytopreparatory method, which may result in a lower threshold to initiate further workup for MTC.

Ectopic Cervical Thyroid Tissue Affected by Fibrosing Hashimoto's Thyroiditis Mimicking Multifocal Metastatic Papillary Thyroid Carcinoma-A Hard Lesson Learnt from an Unusual Case.

We describe a case of ectopic cervical thyroid tissue which was involved by fibrosing Hashimoto's thyroiditis and which mimicked metastatic papillary thyroid carcinoma both on fine needle aspiration cytology and biopsy. The patient underwent total thyroidectomy which revealed fibrosing Hashimoto's thyroiditis, but no carcinoma. The entire thyroidectomy specimen was submitted for histopathological assessment. Even in the resected thyroidectomy specimen, there were cytological changes that were strongly reminiscent of papillary thyroid carcinoma. However, interpreted in the correct clinico-pathological context, these cytological alterations were deemed to be reactive secondary to the fibro-inflammatory process.

Atypia of undetermined significance/follicular lesion of undetermined significance: Asian vs. non-Asian practice, and the Singapore experience.

The Bethesda System for Reporting Thyroid Cytopathology has paved the way for comparisons of the practice of thyroid cytology in many different regions. However, there have been comparatively few studies documenting differences between Asian and non-Asian practice. Here, we aim to compare a few key parameters between the two regions, focusing on the indeterminate category of atypia of undetermined significance (AUS)/follicular lesion of undetermined significance (FLUS). We compared its incidence, resection rates (RRs), risk of malignancy (ROM), rate of repeat fine needle aspiration (rFNA), ROMs of cytomorphologic subcategories of nuclear atypia (AUS-N) vs. architectural atypia (AUS-A), and, finally, the incidence of papillary thyroid carcinoma (PTC) vs. follicular neoplasms (FNs) in resected AUS/FLUS cases in Asian and non-Asian regions. Where possible, these metrics were compared with the Singapore experience from a tertiary referral institution. While the incidence of AUS/FLUS was similar in both regions, we found geographical differences in the RRs and ROMs, which may reflect a higher collective threshold for surgery in Asian countries. However, both cohorts showed higher ROMs in the AUS-N subcategory as compared to the AUS-A subcategory, supporting the subclassification of the AUS/FLUS based on the presence of nuclear atypia. We also observed a higher incidence of AUS-N coupled with a higher incidence of PTC in resected AUS/FLUS nodules in Asian cohorts, while AUS-A and follicular-patterned neoplasms featured more prominently in the non-Asian cohorts. These incidences may account for the starkly different molecular approaches that we noted-in Asian (chiefly Korean and Chinese) centers, BRAF mutational analysis was favored, while gene panels and gene expression classifiers were more frequently applied in non-Asian centers (chiefly in the United States of America). Overall, the data from Singapore appears more closely aligned to non-Asian trends, despite its geographical location in Southeast Asia and its predominantly Asian population. We conclude that there is significant heterogeneity in the outcomes of the AUS/FLUS categories between and within regions, which is only partially explained by regional variations, and may also reflect different regional diagnostic and management practices. This highlights the importance of understanding the local context in the interpretation of indeterminate Bethesda categories, rather than adopting a "one-size fits all" approach.

A common MET polymorphism harnesses HER2 signaling to drive aggressive squamous cell carcinoma.

c-MET receptors are activated in cancers through genomic events like tyrosine kinase domain mutations, juxtamembrane splicing mutation and amplified copy numbers, which can be inhibited by c-MET small molecule inhibitors. Here, we discover that the most common polymorphism known to affect MET gene (N375S), involving the semaphorin domain, confers exquisite binding affinity for HER2 and enables METN375S to interact with HER2 in a ligand-independent fashion. The resultant METN375S/HER2 dimer transduces potent proliferative, pro-invasive and pro-metastatic cues through the HER2 signaling axis to drive aggressive squamous cell carcinomas of the head and neck (HNSCC) and lung (LUSC), and is associated with poor prognosis. Accordingly, HER2 blockers, but not c-MET inhibitors, are paradoxically effective at restraining in vivo and in vitro models expressing METN375S. These results establish METN375S as a biologically distinct and clinically actionable molecular subset of SCCs that are uniquely amenable to HER2 blocking therapies.

The Milan system for reporting salivary gland cytology: A retrospective analysis of 1384 cases in a tertiary Southeast Asian institution.

BACKGROUND: The Milan System for Reporting Salivary Gland Cytopathology (MSRSGC) aims to provide a common language for risk stratification and management. We examine the incidence of MSRSGC categories and the corresponding risk of malignancy (ROM) within a tertiary referral centre in Southeast Asia. METHODS: Salivary gland fine needle aspirations (FNAs) performed within a 10-year period were classified retrospectively according to the MSRSGC. Cytohistologic correlation was performed. The results were compared with the existing literature, including Asian and Western studies. RESULTS: A total of 1384 salivary gland FNAs were evaluated, 421 with corresponding histology. The category distribution was: nondiagnostic, 28.9%; nonneoplastic, 18.0%; atypia of undetermined significance (AUS), 9.8%; benign neoplasm, 32.9%; salivary gland neoplasm of uncertain malignant potential (SUMP), 5.7%; suspicious for malignancy, 1.6%; and malignant, 3.2%. The ROMs were: nondiagnostic, 10.0%; nonneoplastic, 17.5%; AUS, 29.5%; benign neoplasm, 0.5%; SUMP, 17.1%; suspicious for malignancy, 83.3%; and malignant, 100.0%. Our relatively high nondiagnostic rate likely reflects preanalytical factors, whereas our low malignancy rate may be related to population and health care accessibility. Our nonneoplastic ROM was 17.5% compared with 5% to 10% in the literature, likely due to the relatively small number of excised cases; the ROM for SUMP was 17.1% versus 21% to 44% in the literature, possibly reflecting a significant proportion of benign basaloid neoplasms on histology. Interestingly, all false-negative cases in the nonneoplastic category were lymphoid-rich lesions. CONCLUSION: This is one of the largest single-institution studies in the existing literature documenting both the incidence and ROMs of MSRSGC categories. We also highlight specific challenges surrounding lymphoid-rich lesions.

SOX11 expression in a case of papillary thyroid carcinoma with fibromatosis/fasciitis-like stroma containing BRAF c.1799_1801delTGA and CTNNB1 c.133T>C mutations.

We describe a case of papillary thyroid carcinoma with fibromatosis/fasciitis-like stroma (PTC-FLS) that contained the rare BRAF c.1799_1801delTGA (p.V600_K601delinsE) mutation, which has not previously been reported in this tumour, as well as the CTNNB1 c.133T>C (p.S45P) mutation. We also report the novel observation that spindle cells of the mesenchymal component exhibit diffuse nuclear but not cytoplasmic expression of SOX11, whereas the malignant epithelial cells did not. This suggests that immunoreactivity for SOX11 can be an alternative diagnostic tool for evaluating cases of PTC-FLS where the nuclear expression of ß-catenin is ambiguous.

Nanoparticles promote in vivo breast cancer cell intravasation and extravasation by inducing endothelial leakiness.

While most cancer nanomedicine is designed to eliminate cancer, the nanomaterial per se can lead to the formation of micrometre-sized gaps in the blood vessel endothelial walls. Nanomaterials-induced endothelial leakiness (NanoEL) might favour intravasation of surviving cancer cells into the surrounding vasculature and subsequently extravasation, accelerating metastasis. Here, we show that nanoparticles induce endothelial leakiness through disruption of the VE-cadherin-VE-cadherin homophilic interactions at the adherens junction. We show that intravenously injected titanium dioxide, silica and gold nanoparticles significantly accelerate both intravasation and extravasation of breast cancer cells in animal models, increasing the extent of existing metastasis and promoting the appearance of new metastatic sites. Our results add to the understanding of the behaviour of nanoparticles in complex biological systems. The potential for NanoEL needs to be taken into consideration when designing future nanomedicines, especially nanomedicine to treat cancer.

Predictors of thyroxine replacement following hemithyroidectomy in a south east Asian cohort.

BACKGROUND: Thyroxine replacement following a hemithyroidectomy is not commonly discussed during consent for the procedure as the risk of hypothyroidism is perceived to be low. METHODS: Retrospective review of 901 patients who underwent hemithyroidectomy at a tertiary referral institution during the period January 2000 to December 2015. The main outcome studied was the overall incidence of hypothyroidism and the associated risk factors. RESULTS: Hypothyroidism developed in 123 (13%) patients and 94 patients (10%) required hormone supplementation over a mean follow up of 21 months (range 1-168 months). Preoperative TSH of more than 2.5 was seen in 38 of 123 (31%) of patients. Presence of diffuse thyroiditis was the only independent risk factor on multivariate analysis (P = 0.002) found to be associated with the development of hypothyroidism. CONCLUSION: After thyroid lobectomy, approximately one in 10 patients requiring thyroid hormone treatment for hypothyroidism. Presence of diffuse thyroiditis is a significant risk factor for hypothyroidism.

Performance of a Multigene Genomic Classifier in Thyroid Nodules With Indeterminate Cytology: A Prospective Blinded Multicenter Study.

Importance: Approximately 20% of fine-needle aspirations (FNA) of thyroid nodules have indeterminate cytology, most frequently Bethesda category III or IV. Diagnostic surgeries can be avoided for these patients if the nodules are reliably diagnosed as benign without surgery. Objective: To determine the diagnostic accuracy of a multigene classifier (GC) test (ThyroSeq v3) for cytologically indeterminate thyroid nodules. Design, Setting, and Participants: Prospective, blinded cohort study conducted at 10 medical centers, with 782 patients with 1013 nodules enrolled. Eligibility criteria were met in 256 patients with 286 nodules; central pathology review was performed on 274 nodules. Interventions: A total of 286 FNA samples from thyroid nodules underwent molecular analysis using the multigene GC (ThyroSeq v3). Main Outcomes and Measures: The primary outcome was diagnostic accuracy of the test for thyroid nodules with Bethesda III and IV cytology. The secondary outcome was prediction of cancer by specific genetic alterations in Bethesda III to V nodules. Results: Of the 286 cytologically indeterminate nodules, 206 (72%) were benign, 69 (24%) malignant, and 11 (4%) noninvasive follicular thyroid neoplasms with papillary-like nuclei (NIFTP). A total of 257 (90%) nodules (154 Bethesda III, 93 Bethesda IV, and 10 Bethesda V) had informative GC analysis, with 61% classified as negative and 39% as positive. In Bethesda III and IV nodules combined, the test demonstrated a 94% (95% CI, 86%-98%) sensitivity and 82% (95% CI, 75%-87%) specificity. With a cancer/NIFTP prevalence of 28%, the negative predictive value (NPV) was 97% (95% CI, 93%-99%) and the positive predictive value (PPV) was 66% (95% CI, 56%-75%). The observed 3% false-negative rate was similar to that of benign cytology, and the missed cancers were all low-risk tumors. Among nodules testing positive, specific groups of genetic alterations had cancer probabilities varying from 59% to 100%. Conclusions and Relevance: In this prospective, blinded, multicenter study, the multigene GC test demonstrated a high sensitivity/NPV and reasonably high specificity/PPV, which may obviate diagnostic surgery in up to 61% of patients with Bethesda III to IV indeterminate nodules, and up to 82% of all benign nodules with indeterminate cytology. Information on specific genetic alterations obtained from FNA may help inform individualized treatment of patients with a positive test result.